Non-hazardous pest control

ABSTRACT

A pesticide and a method of using the pesticide to kill invertebrates, especially insects, arachnids and larvae. The method is to prepare a mixture of a carrier with the pesticide and apply the mixture to the insects, arachnids and larvae and their habitat. The pesticide is a neurotransmitter affector utilizing octopamine receptor sites in the insects, arachnids and larvae. The affector agent is a chemical having a six membered carbon ring having substituted thereon at least one oxygenated functional group. The affector agent is a chemical component of a plant essential oil and is naturally occurring. Deposition of the chemicals of the present invention on a surface provides residual toxicity for up to 30 days. Various carriers for the affector agent are disclosed. The chemicals of the present invention deter feeding of insects, arachnids and larvae and also retard growth of the larvae of the insects and arachnids.

CROSS REFERENCE TO RELATED APPLICATIONS

The present invention is a divisional application of application Ser.No. 09/056,712, filed Apr. 8, 1998 which issued as U.S. Pat. No.6,183,767B1, which is a divisional application of application Ser. No.08/870,560, filed Jun. 6, 1997 which issued as U.S. Pat. No. 6,004,569,Dec. 21, 1999, which is a continuation-in-part of application Ser. No.08/657,585, filed Jun. 7, 1996, which issued as U.S. Pat. No. 6,114,384,Sep. 5, 2000, which is a continuation-in-part of application Ser. No.08/553,475, filed Nov. 9, 1995, now U.S. Pat. No. 5,693,344, and acontinuation of application PCT/US94/05823, May 20, 1994, which issuedas U.S. Pat. No. 5,693,344, Dec. 2, 1997, which in turn is acontinuation-in-part of U.S. patent application Ser. No. 08/065,594,filed May 21, 1993, which has subsequently issued as U.S. Pat. No.5,439,690, issued Aug. 8, 1995.

BACKGROUND OF THE INVENTION

The present invention relates to a method of controlling pests and moreparticularly to a method of preparing and applying a pesticide whichaffects octopamine receptor sites in insects, arachnids and larvae.

Many chemicals and mixtures have been studied for pesticidal activityfor many years with a goal of obtaining a product which is selective forinvertebrates such as insects, arachnids and larvae thereof and haslittle or no toxicity to vertebrates such as mammals, fish, fowl andother species and does not otherwise persist in and damage theenvironment. Most products of which the applicants are aware and whichhave sufficient pesticidal activity to be of commercial significance,also have toxic or deleterious effects on mammals, fish, fowl or otherspecies which are not the target of the product. For example,organophosphorus compounds and carbamates inhibit the activity ofacetylcholinesterase in insects as well as in all classes of animals.Chlordimeform and related formamidines are known to act on octopaminereceptors of insects but have been removed from the market because ofcardiotoxic potential in vertebrates and carcinogenicity in animals anda varied effect on different insects. Also, very high doses are requiredto be toxic for certain insect species.

It is postulated that amidine compounds affect the octopamine sensitiveadenylate cyclase present in insects [Nathanson et al, Mol. Parmacol20:68-75 (1981) and Nathanson, Mol. Parmacol 28:254-268 (1985)]. Anotherstudy was conducted on octopamine uptake and metabolism in the insectnervous system [Wierenga et al, J Neurochem 54, 479-489 (1990)]. Thesestudies were directed at nitrogen containing compounds which mimic theoctopamine structure.

Insecticides such as trioxabicyclooctanes, dithianes, silatranes,lindane, toxaphen, cyclodienes and picrotoxin act on the GABA (gammaamino butyric acid) receptor. However, these products also affectmammals, birds, fish and other species.

There is a need for a pesticide which targets only insects, arachnidsand their larvae and does not produce unwanted and harmful affects onother species.

BRIEF SUMMARY OF THE INVENTION

It is a primary object of the present invention to provide a method ofpreparing and applying a pesticide which kills invertebrates, especiallyinsects, arachnids and their larvae and has no harmful effects on otherspecies including mammals, fish and fowl.

It is a further object of the present invention to provide a method ofpreparing and applying a pesticide which exerts its pesticidalproperties through the octopamine receptor site in insects, arachnidsand their larvae and other invertebrates.

It is still another object of the present invention to provide a methodof preparing and applying a pesticide at relatively low concentrationswhich will be effective over a comparatively long period of time such asat least 24 hours.

In accordance with the teachings of the present invention, there isdisclosed a method of killing insects and arachnids and larvae thereof.The steps include preparing a mixture of a carrier with an affectoragent which interferes with the neurotransmitters of the octopaminereceptor site in insects, arachnids and their larvae and applying themixture to insects, arachnids, larvae and their habitat. The affectoragent interacts with octopamine receptor sites in the insects, arachnidsand larvae and interferes with neurotransmission in the invertebrate butdoes not affect mammals, fish and fowl. The agent is a chemical havingthe structure of a six member carbon ring, the carbon ring havingsubstituted thereon at least one oxygenated functional group.

There is further disclosed a method of killing insects and arachnids andlarvae thereof. A blend of cinnamic alcohol, eugenol and alpha terpineolis prepared. The blend is mixed with a carrier to produce a uniformmixture. The mixture is applied to insects and arachnids and larvae andtheir habitat. The blend interacts with octopamine receptor sites in theinvertebrate and interferes with neurotransmission in the invertebratebut does not affect mammals, fish and fowl.

In another aspect, there is disclosed a pesticide which has an affectoragent having a six member carbon ring. The carbon ring has substitutedthereon at least one oxygenated functional group. The affector agentaffects the octopamine receptor site in invertebrates including insects,arachnids and their larvae. The affector agent is intimately mixed witha carrier. Exposure of insects, arachnids and their larvae to theaffector agent produces a disruption of the octopamine receptor site inthe invertebrates to interfere with neurotransmission in theinvertebrate and the death of the exposed invertebrate.

Further disclosed is a method of killing insects, arachnids, and larvae.A mixture is prepared of a chemical derived from a plant essential oilwith a carrier. The chemical has at least one oxygenated functionalgroup therein. The chemical has octopamine receptor site inhibitoryactivity. The mixture is applied to insects, arachnids, larvae and theirhabitat. The chemical interacts with an octopamine receptor site in theinsects, arachnids and larvae and interferes with neurotransmission inthe insects, arachnids and larvae thereof but does not affect mammals,fish and fowl.

In another aspect there is disclosed a method of controlling insects,arachnids and their larvae. An emulsion is prepared of an affector agentwhich disrupts neurotransmission at the octopamine receptor site ininsects, arachnids and their larvae. The mixture is applied to insects,arachnids, larvae and their habitat. The agent interacts with octopaminereceptor sites in the insects, arachnids and larvae and deters thefeeding of the insects, arachnids and larvae but does not affectmammals, fish and fowl.

In yet another aspect there is disclosed a method of controllinginsects, arachnids and larvae. An affector agent mixed with a carrier isapplied to larvae of the insects and arachnids and their habitat. Theaffector agent retards the growth of the larvae. The affector agentinteracts with octopamine receptor sites in the larvae of the insectsand arachnids and interferes with neurotransmission in the larvae butdoes not affect mammals, fish and fowl. The affector agent is anaturally occurring organic chemical having at least six (6) carbonatoms.

In addition, there is disclosed a method of killing insects, arachnidsand larvae thereof. A mixture is prepared of a carrier and a naturallyoccurring organic chemical having at least six carbon atoms. Thechemical has octopamine receptor site inhibitory activity. The mixtureis applied to insects and arachnids and larvae thereof and theirhabitat. The chemical interacts with an octopamine receptor site in theinsects and arachnids and larvae thereof and interferes withneurotransmission in the insects, arachnids and larvae thereof but desnot affect mammals, fish and fowl.

These and other objects of the present invention will become apparentfrom a reading of the following specification, taken in conjunction withthe enclosed drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the effect of the 3-B mixture of the presentinvention on the intracellular concentration of [Ca²⁺] in neuronalcells.

FIG. 2 is a chart showing the contraction of cockroach leg musclesinduced by electrical stimulation when the 3-B mixture of the presentinvention is injected into the thorax of a live cockroach.

FIG. 3 is a chart showing the contraction of cockroach leg muscles in anisolated leg induced by electrical stimulation when the 3-B mixture ofthe present invention is applied to the isolated leg.

FIG. 4 is a chart showing the test of FIG. 2 using acetone as a controlwithout the 3-B mixture.

FIG. 5 is a chart showing the test of FIG. 3 using acetone as a controlwithout the 3-B mixture.

FIG. 6 is a series of charts showing transmission of signals from themechanoreceptors of the cerci after application of the 3-B mixture and acontrol to the abdominal nerve of the cockroach.

FIG. 7 is a chart showing feeding damage to cabbage plant leaf discswith the 3-B mixture as compared to a control.

DESCRIPTION

Physiological activity to invertebrate insects, arachnids and theirlarvae are produced by the chemicals of the present invention and bymixtures of these chemicals. The following chemicals having a six membercarbon ring and having substituted thereon at least one oxygenatedfunctional group are representative of the chemicals of the presentinvention but these are not to be considered as being the totality ofchemicals and are not a limitation.

The above-listed materials are all components of plant essential oils. Afurther plant essential oil which is a chemical of the present inventionis

Anethole, carvacrol, citronellal, eugenol, D-pulegone, alpha-terpineoland thymol are all monoterpenes each having ten (10) carbon atomstherein.

The present invention is not limited to the chemicals listed herein.

All of the chemicals of the present invention are naturally occurringorganic chemicals which are devoid of halogens. Further, several of thechemicals of the present invention are considered by the U.S.Environmental Protection Agency (EPA) to be safe for humans and exemptedfrom registration. Thus, these chemicals do not require prior approvalor registration with the EPA. Some of these chemicals have been added toprior art insecticides as attractants and repellents. However, therehave been no reports of these chemicals having toxic pesticidal activityat the concentrations disclosed in the present invention.

All of the chemicals of the present invention act as agonists orantagonists on the octopamine receptor site in insects, arachnids andtheir larvae and, consequently, produce physiological effects in exposedinvertebrates. The chemicals of the present invention are considered tobe affector agents. Exposure to reduced concentrations of the chemicalsor exposure for brief times affect the feeding habits of the exposedinsects, arachnids and larvae. This is important in those invertebrateswhich feed on vegetation, since there is reduced damage to plants byinvertebrates which have received sublethal concentrations of thechemicals of the present invention. Also, the insects, arachnids orlarvae which survive exposure to the chemicals of the present inventionare shown to be stunted in growth.

Alpha-terpineol, eugenol and cinnamic alcohol were dissolved in acetoneand designated as sample 3B. The range of weight percentages of thecomponents of the blend are alpha-terpineol 10%-50%, eugenol 10%-50% andcinnamic alcohol 20%-35%. The preferred blend has equal parts by weightof each of the components. Male and female American cockroaches wereinjected with 3B into the abdomen. Overt signs of toxicity were observedat 1 mg/roach in 2 μl of acetone. At lower doses no symptoms wereobserved. By this approach, 2 out of 6 died within 30 minutes. For thosesurviving, some showed locomotive difficulties. No hyperactivity wasobserved, even in those that died quickly. In come cases, (2/6) treatedinsects died after 2 to 3 days.

None of the American cockroaches died when they were treated throughtopical application prior to 24 hours. A 40% (4/10) mortality wasobserved 24 hours after treatment and this effect was time dependent.After 72 hours, 100% died. Control insects receiving 2 μl of onlyacetone through injection showed no ill effects. (Table 1).

TABLE 1 Time course effect of 3-B (1 mg/ insect) on American cockroachestreated by topical application. Time (hr.) Mortality-3BMortality-Control 24 4/10 0 48 6/10 0 72 10/10  0

The data from the above study suggests that the action of 3B depends onthe site of application, i.e., abdominal injection vs. topicalapplication for the whole body.

3B was applied to different areas of the insect. When given to theventral sternum region (at the base of the hind legs) of the Americancockroach, 3B was most toxic at 125 μg/insect. In that case, overt toxicsigns were observed within 10 minutes. A lethal dose of 250-500μg/insect was injected and the insects died within 30 minutes (Table 2).

TABLE 2 Time-course and dose-response of 3B on American cockroachestreated by injection through the ventral stenum. Mortality Time TestDoses μg/insect (hrs.) 50 100 125 250 500 0.5 0 2/10 6/10 10/10 10/101.0 2/10 3/10 7/10 — — 1.5 5/10 7/10 9/10 — — 2.0 5/10 9/10 10/10  — —3.0 6/10 9/10 — — — 5.0 8/10 10/10  — — — 24.0 10/10  — — — — *Nomortality was found in insects treated exactly in the same manner withequal volume of acetone (vehicle) alone as control.

In the latter case of injection of a lethal dose the hind legs appearedto be paralyzed and the fore and mid legs moved rapidly, although therewas no overt hyperexcitation. This phenomenon of quick death was alsoobserved in German cockroaches that were treated through topicalapplication (3B is more toxic to German than to American cockroaches).At 125 μg/insect, 80% (8/10) of German cockroaches were knocked down in2 to 3 hours and died within 24 hours (dose was 125 μg 3B per insectwith 0.4 μl acetone).

The above data support the observation that site of application givesvarying degrees of toxicity. For further confirmation, 1 mg 3B in 20 mlacetone was applied to small jars and all surfaces were covered. Onehour after the acetone had complete evaporated, 5 American cockroacheswere introduced to each jar. Control jars were treated exactly as abovebut the 20 ml acetone only. All cockroaches died within 10 to 30 minutesin the jars containing 3B. None died in the control jars. Some insects(3/5) showed hyperactivity within 1 to 3 minutes after exposure. At 8minutes, hind leg paralysis was observed. These “walk across” data areconsistent with the previous study in which a rapid death was observedwhen 3B was given to the ventral sternum region rather than the abdomen.The fact that the toxicants might penetrate faster through the legs (nochitin layer as in the body) support the notion that thepermeability/penetration of 3B plays a key role in its toxicity (Table3).

TABLE 3 Time-course effect of 3B (1 mg/jar) on American cockroachesexposed to pre-treated surface one hour after 3-B application. Time(min.) Mortality 10 3/5 15 3/5 20 4/5 30 5/5 *No death in insectsexposed to surface treated with acetone alone.

Based on these data, the same treated jars were used for a residualstudy. In this experiment, American cockroaches were transferred to thetreated and control jars at different times from the point in time atwhich acetone evaporated, i.e., 24, 48, 96, 72 hours and 7 days.Interestingly, all insects died after being exposed to treated jars,even 7 days after 3B application. However, it took longer for theroaches to die in proportion to the length of time from initial 3Bapplication to the jar. Some toxic signs were observed in all casesafter exposure. Further, when the same experiment was repeated withGerman cockroaches, greater toxic effects and more rapid effects wereobserved. It appears that some degradation occurs in toxic effects withtime (Table 4).

TABLE 4 Effect of 3-B (1 mg/jar) when applied to surfaces on Americancockroaches exposed to these surfaces at different times after surfacetreatment. Time elapsed after Time required to kill 3-B application 100%of insects (days) (days) 1 1 2 3 3 6 5 8  7* 10 (6/10 died) *Only 6insects died when 10 cockroaches were exposed to pretreated jars 7 daysafter 3-B application.

The toxic effects shown in Table 4 could be protracted by combining oilswith different characteristics. Eugenol, one of the ingredients of 3-B,was dissolved in galoxolide, a perfume oil that imparted longer lastingproperties related to evaporation and oxidation to the pesticideproperties of eugenol. Mixtures having 30%-60% galoxolide and 40%-70%eugenol by weight have been effective. As an example, eugenol andgaloxolide were mixed in equal parts, 1 mg each, in 10 ml of acetone andagitated vigorously for two minutes and then applied (1 mg ofmixture/jar) as above to small jars and all surfaces covered. The dataof this example (Table 5) show that the lethal effect of the mixture wasextended and enhanced.

TABLE 5 Time elapsed after Time required to eugenol/galoxolide*application kill 100% of insects (weeks) (days) 1 1 2 1 3 1 4 1 *1, 3,4, 6, 7, 8-hexahydro-4, 6, 6, 7, 8,8-hexamethyl-cyclopenta-gamma-2-benzopyran

Cockroach abdominal nerve cord showed the second highest uptake ofoctopamine of all tissues studied [J. Neurochem 54 479-489 (1990)]. Thehigh efficacy of the chemicals of the present invention in the “walkacross” study is attributed to the high concentration of octopaminereceptors on the ventral nerve cord in close proximity to the hind legs.

Since the toxic signs produced by 3B did not indicate cholinergic actionpatterns, other major possibilities such as GABA receptor, octopaminereceptor/biogenic amine binding, [Ca²+], or mitochondrial respiratorypoison were considered:

1. GABA receptor-chloride channel study: This site of action is known tobe one of the major action sites for a number of insecticides. When theaction of 3B was tested, 3H-EBOB ([³H]n—propyl bicyclo ortho benzoate)and 35S-TBPS (bicyclo phosphorous esters) were used. The former ligandwas used based on the finding that EBOB has been shown to be both ahighly toxic and high affinity radioligand for the GABA-receptorconvulsant binding site in insects. It also shows identical oroverlapping binding sites with seven classes of insecticides:trioxabicyclooctanes, dithianes, silatranes, lindane, toxaphen,cyclodienes and picrotoxinin. The TBPS has been hindered by poortoxicological relevance and binding affinity in insects. Bothradioligands were used in this study for comparison. As shown in Table6, 3B induced no antagonistic action on the binding affinity of 3H-EBOBor 35S-TBPS at concentrations of 3B ranging from 10 μM to 10 NM. Only at100 μM was a significant effect seen. However, at such a highconcentration (100 μM), the specificity for the site of action isunlikely. In contrast, heptachlorepoxide alone and a mixture ofEndosulfan I (60%) and Endosulfan II (40%) as a positive standard werehighly active even at 10 Nm. These data indicate that this assay systemworks poorly with 3B, and that the lack of its action is due to itsinaccessibility to the GABA receptor site. The fact that the cyclodieneresistant London strain showed cross-resistance to 3B supports the abovedata and rules out GABA receptor as the target or site of action for 3B.

TABLE 6 Effect of 3B on GABA-receptor binding. dpm per 200 μgsynaptosomal membrane protein (X ± SD) Tested Concentration μM ³H-EBOB³⁵S-TBPS 3-B (μM) 0 2558 ± 159 3361 ± 297 0.01 2069 ± 98 2991 ± 101 0.102069 ± 198 2939 ± 111 1.0 2088 ± 76 2917 ± 85 10 2111 ± 151 3001 ± 173100 2109 ± 88 2985 ± 203 Endosultan Mix μM 0 2558 ± 159 3361 ± 297 0.011886 ± 71 2477 ± 162 0.1 1009 ± 83 1358 ± 101 1.0  350 ± 35  552 ± 43 10 255 ± 29  360 ± 15 100  241 ± 18  285 ± 11 Hepatachlorepoxide μM 0.0012010 ± 91 2470 ± 188 0.01 1583 ± 77 2000 ± 75 0.10 1221 ± 63 1699 ± 1091.0 1142 ± 85 1493 ± 99 10  591 ± 41  685 ± 66 Unlabeled Ligand μM 0.001 499 ± 34  533 ± 25 0.01  346 ± 29  381 ± 17 0.10  206 ± 15  211 ± 19

Control value (solvent alone) was 2558±159X3361±297 for EBOB X TBPS,respectively.

2. Octopamine receptor/biogenic amine binding site: Biogenic amines areknown to carry out a number of physiological functions through theirspecific receptors in insects. The octopamine receptor is the mostdominant biogenic amine receptor in insects. Certain acaricides, such achlordimeform, are known to act on octopamine receptors, causing avariety of symptoms, including behavioral changes. When 3B was incubateddirectly with a homogenate of the nerve cord of American cockroaches, asignificant increase in cylical AMP (cAMP) was found at a dosage of 1μM. The chemical octopamine was used as a positive control and induced asignificant increase in cAMP at 1 μM. Additional evidence thatoctopamine is the main site of action of 3B is that in co-treatment of3B and octopamine, 3B abolished the octopamine-induced increase in cAMP.

To confirm that 3B is an octopamine receptor toxicant, two importantbiomarkers were measured: heart beats/30 seconds and cAMP—dependentprotein kinase (PKA) activity. These two are considered particularlyimportant in identifying octopamine receptor activity as the cockroachheart has been shown to have a high concentration of octopaminereceptors. When a 200 μg/insect concentration of 3B was applied to thesternum region of the alive and intact American cockroach, a significantincrease was seen in heart beats/30 seconds and this was accompaniedwith an increase in cAMP. As before, higher concentrations of 3Bresulted in a decrease in heart beats (Table 7).

TABLE 7 Effect of 3-B on Arnerican cockroach heart beats/30 seconds.Tested Doses, μg Before Treatment 30 Min. After Treatment control 55 ±1.9 55 ± 3.2 200 55 ± 2.1 71 ± 4.5 300 58 ± 0.81 67 ± 1.63 600 57 ± 0.4741 ± 2.4 900 53 ± 1.9 38 ± 2.8 octopamine (20 μg) 54 ± 2.1 75 ± 3.6chlordimeform (20 μg) 51 ± 1.4 69 ± 2.5 (positive control)

In addition, when 3B was incubated with synaptasomal preparation fromAmerican cockroach heads, a significant increase in PKA activity wasfound, which is consistent with the above conclusion (Table 7).

TABLE 8 In Vivo effect to 3-B (200 μg/roach) on the activity of PKA insynaptosomal membrane of American cockroaches dpm/1 nmol of Kemptide/5min. X ± SD Control 1753 ± 57 3-B 4008 ± 201

3. Possible role of [Ca²⁺]: Because of the locomotive difficultiesnoticed among cockroaches treated with 3B at the ventral sternum region,it was postulated that increases in intramuscular levels of Ca²⁺ couldbe involved in the contractions of hind legs. For this purpose,mammalian cell line PC12 rat chromaffin adrenal cells was used. Thiscell line is known to mimic neuronal cells, particularlycatecholaminergic neurons and has been used as a model for Ca²⁺ inducedpresynaptic transmitter release phenomena. As shown in FIG. 1 when 3Bwas added at 100 μM to these cells (at A), there was no change inintracellular free Ca²⁺. [Ca²⁺] concentration inside PC12 cells wasfound using spectrofluorometric measurements with Fura 2/AM (a cellpenetrating fluorescence probe for free Ca²⁺). A standard positivecontrol, thapsigargin clearly increased the [Ca²⁺]i (at B) even at 500Nm. Also, 10 μM ionomycin, a Ca²⁺-ionophore (at C) produced the expectedincrease of Ca²⁺ entry. These results showed that 3B shows no ability toregulate any type of calcium homeostasis in mammalian cells.

The effect of the chemicals of the present invention on the rat braintissue cells was as follows where cyclical AMP generation is measured asdpm/mg protein

control 5395 ± 43 3-B 5411 ± 391 terpineol 5399 ± 219 eugenol 5461 ± 488phenyl ethyl alcohol 5499 ± 415

Thus, no change was produced in the neurotransmission system of amammal. These data confirm the lack of neurotoxicity of these essentialoils in mammals.

4. Mitochondrial/respiratory poison: Another possible mode of action isthat of mitochondrial or respiratory poisoning. It has been observedthat all mitcohondrial poisons induce hyperactivity at some point intheir action. However, when American cockroaches were treated with atopical application of 3B, no hyperactivity or hyper excitation wasobserved at any stage of poisoning at all concentrations used. On theother hand, when American cockroaches were exposed to pre-coated jarswith 1 mg of a 60% solution of 3B, the insects showed hyper excitation.These data suggest that the mode of entry is a determining factor in themode of action of these essential oils. These observations support theidea that 3B is not a type of poison that attacks the Na⁺channel as amain target. The fact that the Kdr-resistant strain (with a mutated Na⁺channel making it insensitive to DDT and pyrethroids) did not showcross-resistance to 3B also supports this diagnosis.

Under the above circumstances, where all of the known major sites ofaction for insecticides were found to be insensitive to 3B, except theantagonistic effect on the octopamine receptor, the possibility wasconsidered that the mode of action of this group of chemicals is totallynovel.

It was noted that the toxicity of 3B varies according to the site ofapplication, at least in the case of the American cockroach. Such anobservation indicates that 3B is likely to be not so systemic in action,and if one site produces a high toxicity, its target is likely to belocated very close to that site of application. In view of thelocomotive difficulty observed in American cockroaches treated at thecentral sternum, it was reasoned that the thoracic ganglia could beaffected. When 3B (250 mg. in 0.4 μl acetone) was injected into thethorax of an alive cockroach and its hind leg contractions were inducedby an external electrical stimulus applied to the outside of the body,the ability of the leg muscles to respond to stimuli totally disappearedwithin one minute (FIG. 2). When 3-B was applied to the leg in isolationby directly giving electrical stimuli, no effect of 3B was found (FIG.3). These data suggest that the effect of 3B is on the nervous systemand not on the muscles. Control tests using acetone (0.4 μl) without any3-B showed no effects (FIGS. 4 and 5). This possibility was furthertested using the 6^(th) abdominal ganglion and studying transmission ofsignals from the mechanoreceptors of the cerci generated by air puffing.For this purpose, the abdominal cavity was opened, exposing the entireabdominal nerve cord. A suspension solution of 3B in 200 μl insectsaline was directly applied to the entire abdominal nerve cord. Theresults were drastic, producing complete blockage of transmission at250-500 PPM within 5 minutes from the time of application (FIG. 6). Evenat 10 PPM the blocking effect of 3B was apparent, producing visibleeffects in 15 minutes. These results clearly indicate that 3B is anonsystemic nerve blocker.

A further study was conducted on the cockroach to determine the effectof other chemicals of the present invention on the heart rate of theinsects. The experiment conditions were as previously described inapplying 300 μg of the respective chemical to the entire abdominal nervecord. Each insect group was comprised of three individuals. All heartrates were observed and counts were recorded three times for eachindividual, i.e., nine counts before introduction of the chemical, andnine counts 30 minutes after application of approximately 300 μg ofchemical. The data from these heart beat tests agreed with thepreviously described “walk-across” test. The following chemicals showedmeasurable change in heart rate thirty (30) minutes after application ofthe chemical: terpineol, eugenol, phenyl ethyl alcohol, benzyl acetateand benzyl alcohol.

Also the following mixtures showed measurable change in heart rate:terpineol with eugenol, terpineol with phenyl ethyl alcohol and eugenolwith phenyl ethyl alcohol.

The toxicity of the individual chemicals was also determined by topicalapplication to early 4^(th) instar Asian armyworms (Spodoptera litura)(15-20 mg live wt) and measurement after 24 hours (Table 9).

TABLE 9 Toxicity of chemicals of the present invention. 95% con- 95%con- LD50 fidence LD90 fidence Compound (μg/larva) interval (μg/larva)interval α-terpineol 156.0 148.7-163.7 206.4 190.4-249.9 eugenol 157.7149.9-165.8 213.0 194.8-263.3 cinnamic LD50 > 250 alcohol (+)-terpinen-130.4 121.8-139.5 205.8 180.2-283.8 4-ol (−)-terpinen- 122.9 108.1-139.7276.0 202.4-583.9 4-ol carvacrol 42.7 37.7-48.3 73.8 55.7-142.0 Dpulegone 51.6 49.0-54.4 69.7 62.3-91.3 t-anethole 65.5 61.7-69.6 98.888.4-129.1 thymol 25.5 22.9-28.3 46.8 38.5-74.5 citronellal 111.3103.9-119.1 153.4 130.8-223.5

In establishing the LD50 dose, it was noted that all larvae treated withpulegone, even at the lowest doses tested, were almost immediatelyparalyzed. However, at lower doses, many or most of these larvaeovercame this effect, whereas at higher doses larvae succumbed. Todetermine if the observed sublethal toxicity resulted in any long-termeffect, subsequent growth of larvae treated at the three lowest doses(20, 31 and 49 μg per larvae) was monitored and compared this to growthof control (untreated larvae) at 72 and 100 hours after treatment (Table10).

TABLE 10 Growth of larvae exposed to chemicals of the present invention.Control 20 μg/larva 31 μg/larva 49 μg/larva Mortality (5) 0 0 2 52 Livewt. - 72 hr. 148 mg. 109 81 52 Live wt. - 110 hr. 363 mg. 289 248 163

The results indicate that subsequent larval growth of survivors of thehighest dose is dramatically retarded. More importantly, the effect isalso seen (and significantly so) at the two lowest doses, whichthemselves produced almost no mortality. Therefore, exposure to evensublethal doses can have significant consequences for larvae.

Dry powder formulations were prepared using the chemicals of the presentinvention. The examples listed below are for the 3B mixture aspreviously described (cinnamic alcohol, eugenol and alpha terpineol).However, these examples are for illustrative purposes only and do notlimit the range of active chemicals. Other mixtures and individualchemicals may also be used. Suggested mixture are phenyl ethyl alcohol,benzyl alcohol, eugenol and alpha terpineol (3C) and benzyl acetate,benzyl alcohol, phenyl ethyl alcohol, cinnamic alcohol and alphaterpineol (3D). The mixtures listed herein are not limiting but aretypical and the present invention is not limited to these mixtures.

The procedure was to place the powder components into a 500 ml dish andapply the chemical (usually a liquid) on the powder. The powder andchemical mixed in the container were placed on an electric tumbler todry for approximately 30 minutes. Approximately 1 cc of the resultingdry powder was applied to a Whatman No. 1 filter paper in a 9 cm Petridish. The dust was spread evenly with a camel hair brush. A control wasused which consisted only of the powder components without the chemicalof the present invention.

The following compositions were prepared: (Tables 12a-12c)

TABLE 12a % by weight amount components 40 20 gr diatomaceous earth 2010 gr calcium carbonate 20 10 gr sodium bicarbonate 10 5 gr Hi-Sil 23310 5 gr active ingredient*

TABLE 12b % by weight amount components 40 20 gr diatomaceous earth 2311.5 gr calcium carbonate 20 10 gr sodium bicarbonate 10 5 gr Hi-Sil 233 7 3.5 gr active ingredient*

TABLE 12c % by weight amount components 40 20 gr diatomaceous earth 2512.5 gr calcium carbonate 20 10 gr sodium bicarbonate 10 5 gr Hi-Sil 233 5 2.5 gr active ingredient* *active ingredient was a mixture of thechemicals of the present invention.

The mixtures were tested by placing ten (10) common fire ants withinrespective Petri dishes which were then covered. The time forirreversible knockdown (KD) to occur (KT) was determined from periodicirregular observations. The insects were considered KD when they were ontheir back, or could be turned onto their back and could not rightthemselves within at least two (2) minutes. KT-50 and KT-90 (time for50% and 90% KD respectively) were calculated by interpolation of KDbetween times when data was collected. The 10% mixture (Table 12a) had aKT 50% of 5 min. 50 sec. and a KT 90% of 6 min 40 sec. For the 7%mixture (Table 12b) KT 50% was 3 min. 40 sec. and KT 90% was 4 min. 40sec. The 5% mixture (Table 12c) had KT 50% of 2 min. 33 sec. and KT 90%of 3 min. 45 sec.

The above data are examples which clearly demonstrate the effectivenessof the mixture of chemicals over concentrations ranging from 5%-10% byweight of the active ingredients. These are typical examples, but arenot limiting. Concentrations as low as 0.1% by weight have also beenshown to be effective for some individual chemicals and for variousmixtures of chemicals. Also much higher concentrations can be used.

As an example of a high concentration, a emulsifiable concentrate hasthe following formulation:

Ingredient Purpose wt. % 3B active ingredient 90.0 Sodium dodecylbenzene sulfonate emulsifier 6.6 Sodium C12-15 Pareth-3 sulfonateemulsifier 2.0 POE 20 Sorbitan Monooleate emulsifier 1.4

Another emulsifiable concentrate is:

Ingredient Purpose wt. % 3B active ingredient 90.0 Caster oil (40 molEO) emulsifier 10.0

The concentrate is diluted with up to 50 to 70 parts of water to 1 partof concentrate to provide an effective aqueous medium. Although theseexamples are for the 3B and 3C mixtures, the formulation is not solimited and an emulsifiable concentrate may be prepared from any of theindividual chemicals of the present invention or any combination ofmixtures of the individual chemicals.

The emulsifiable concentrate 3B mixture was diluted in tap water andsprayed onto cabbage and bean plants to assess efficacy against Asianarmyworms and two-spotted spider mites (Tetranychus urticae)respectively. All values are based on a minimum of four doses with fivereplicates and ten armyworms or thirty adult and/or deutonymph mites perreplicate. Values reported represent the ratios of water to formulation(i.e., dilution rate) (e.g., LD50 of 46.8 means a ratio of water toemulsifiable concentrate of 46.8:1). Mortality was assessed at 24 hours(Table 13).

TABLE 13 Mortality of emulsifiable mixture of 3-B mixture 3-day oldAsian armyworm LD50 (95% C.I.) = 46.8 (44.0-49.8) LD90 (95% C.I.) = 30.6(27.2-40.7) 5-day old Asian armyworm LD50 (95% C.I.) = 53.0 (49.6-56.7)LD90 (95% C.I.) = 33.7 (29.7-45.6) two-spotted spider mite LD50 (95%C.I.) = 82.3 (76.5-88.5) LD90 (95% C.I.) = 55.9 (49.3-75.5)

The diluted emulsifiable concentrate 3B formulation shows good efficacyagainst both ages of armyworms, and even better efficacy against spidermites. Five-day old armyworms are approximately the same size as mature(last instar) diamondback moth larvae, a potential target species. Alsoof importance, it was observed that sublethal concentrations clearlydeter feeding and therefore plant damage. At a dilution of 50:1, 60:1and 70:1, larval mortality is only 64%, 42% and 12% respectively.However, there is minimal feeding damage from surviving larvae. As shownin FIG. 7, discs of cabbage plant leaves with 3-B emulsifiableconcentrate diluted 50:1, 60:1 and 70:1 have areas consumed (ac) after24 hours of less than 5%-30% as compared to a control (without 3-B) ofgreater than 80% area consumed.

Another example of a high concentration is a wettable powder having thefollowing formulation:

Ingredient Purpose wt. % 3B active ingredient 50.0 Silica, hydrated,amorphous absorbent 41.5 Sodium alkyl naphthalene sulfonate dispersant3.0 Sodium dioctyl sulfosuccinate wetting agent 0.5 Sodium dodecylbenzene sulfonate emulsifier 5.0

One part of this powder is mixed with up to 30-50 parts of water toprovide an effective pesticide. This example is for the 3B mixture butis not limited and may be prepared with any of the individual chemicalsof the present invention or any combination of mixtures of theindividual chemicals.

A waterproof dust may be prepared using any of the individual chemicalsor a mixture of any of the chemicals of the present invention. Thefollowing formulation is representative in which the mixture 3C is equalparts by weight of benzyl alcohol, phenyl ethyl alcohol and terpineol:

Ingredient Purpose wt. % 3C active ingredient 5.0 Diatomaceous earthbulking agent 75.0 Hydrophobic silica absorbent 20.0

The following formulations represent two ready to use spraysrepresentative of mixtures 3B and 3C but may be used for other mixturesand for individual chemicals of the present invention:

Ingredient Purpose wt. % 3B active ingredient 5.0 Caster oil (40 mol EO)emulsifier 2.7 POE 20 Sorbitan Monooleate emulsifier 0.6 Isopropylaminealkyl benzene emulsifier 1.7 sulfonate Water diluent 90.0 3C activeingredient 5.0 Caster oil (40 mol EO) emulsifier 2.7 POE 20 SorbitanMoncoleate emulsifier 0.6 Isopropylamine alkyl benzene emulsifier 1.7sulfonate Water diluent 90.0

The individual chemicals of the present invention and mixtures may alsobe used as an aerosol spray. While not limited thereto, the followingformulation is typical:

Ingredient Purpose wt. % 3C active ingredient 5.0 Propanol solubilizingagent 1.5 Carbon dioxide propellant 3.5 Isoparaffinic hydrocarbonsolvent 90.0

A pesticidal shampoo has the following composition where the activeingredient is an individual chemical of the present invention or mixtureof chemicals:

Ingredient wt. % Active ingredient 0.5-10.0 Sequestrant for hard water0.5-3.0 Emulsifier(s) 1.0-5.0 Thickener(s) 0.5-5.0 Foam stabilizer0.5-2.0 Detergent 5.0-20.0 Buffer 0.1-2.0 (to required pH) Dye/colorant0.01 Preservative 0.01-1.0 Deionized water to 100%

Gel formulations have been prepared as follows:

Blue Color 3.00% Active ingredient 15.00% Ethanol 0.42% Carbopol 9340.42%-10% Sodium hydroxide in water 81.16% Water Yellow Color 3.00%Active ingredient 30.00% Ethanol 0.42% Carbopol 934 0.42%-10% Sodiumhydroxide in water 66.16% Water

The active ingredient is an individual chemical of the present inventionor a mixture thereof.

A dry powder formulation consists of mixing an alkaline earth metalcarbonate, such as calcium carbonate, an alkali metal bicarbonate, suchas sodium bicarbonate, the active ingredient, an absorbent material,such as diatomaceous earth and a bulk agent such as HiSil 233.

The relative concentrations of the mixture are preferably about 20%-30%alkaline earth metal carbonate, 15%-25% alkali metal bicarbonate,0.1%-5% active ingredient, 30%-50% absorbent material and 5%-15% bulkagent (all by weight). The granules of powder preferably are ground to asize under 100 microns.

The active ingredients may be individual chemicals of the presentinvention or mixtures thereof.

In all of the above recited examples, it may be desirable to add a traceamount (less than 2%) of a material to provide a pleasant odor, not onlyfor aesthetic reasons but also to identify the areas of a building whichhave been treated. The pleasant odor may be vanilla, cinnamon, floral orother odors which are acceptable to consumers. The pleasant odors arenot limited to the examples given herein.

Obviously, many modifications may be made without departing from thebasic spirit of the present invention. Accordingly, it will beappreciated by those skilled in the art that within the scope of theappended claims, the invention may be practiced other than has beenspecifically described herein.

What is claimed is:
 1. A method of killing Asian army worm larvaecomprising the steps of: preparing d-pulegone in a carrier, applying thed-pulegone in the carrier to the Asian army worm larvae and the habitatthereof wherein the Asian army worm larvae are exposed to an effectivedosage to kill the Asian army worm larvae.
 2. The method of claim 1,wherein the dosage is at least 62.3 μg of d-pulegone per larva.
 3. Themethod of claim 2, wherein 90% of the Asian army worm larvae are killed.4. A method of killing Asian army worm larvae comprising the steps of:preparing d-pulegone in a carrier, applying the d-pulegone in thecarrier to the Asian army worm larvae and the habitat thereof whereinthe effective dosage to kill the Asian army worm larvae is approximately62.3 μg of d-pulegone per larva.
 5. A method of killing Asian army wormlarvae comprising the steps of: preparing d-pulegone in a carrier,applying the d-pulegone in the carrier to the Asian army worm larvae andthe habitat thereof wherein the Asian army worm larvae are exposed to aneffective dosage of at least 49.0 μg of d-pulegone per larva and 50% ofthe Asian army worm larvae are killed.